key: cord-0820360-fwquzrmo authors: Mulu, A.; Alemayehu, D. H.; Alemu, F.; Tefera, D. A.; Wolde, S.; Aseffa, G.; Seyoum, T.; Habtamu, M.; Abdissa, A.; Bayih, A. G.; Beyene, G. T. title: Evaluation of Sample Pooling for Screening of SARS-CoV-2 date: 2020-06-12 journal: nan DOI: 10.1101/2020.06.10.20123398 sha: 9d158bda7c04ec79e5a4dffda7e82a983d40eca6 doc_id: 820360 cord_uid: fwquzrmo Background: The coronavirus disease (COVID-19) pandemic has revealed the global public health importance of robust diagnostic testing. To overcome the challenge of nucleic acid (NA) extraction and testing kit availability efficient method is urgently needed. Objectives: To establish an efficient, time and resource-saving and cost-effective methods, and to propose an ad hoc pooling approach for mass screening of SARS-CoV-2 Methods: Direct clinical sample and NA pooling approach was used for the standard reverse transcriptase polymerase chain reaction (RT-PCR) test of the SARS CoV-2 targeting the envelop (E) and open reading frame (ORF1ab) genomic region of the virus. In this approach, experimental pools were created using SARS CoV-2 positive clinical samples spiked with up to 9 negative samples prior to NA extraction step to have a final extraction volume of 200L (maximum dilution factor of 10). Viral NA was also subsequently extracted from each pool and tested using the SARS CoV-2 RT-PCR assay. Results: We found that a single positive sample can be amplified and detected in pools of up to 7 samples depending on the ct value of the original sample, corresponding to high, medium, and low SARS CoV-2 viral copies/reaction. However, to minimize false negativity of the assay with pooling strategies and with unknown false negativity rate of the assay under validation, we recommend poling of 4 in 1 using the standard protocols of the assay, reagents and equipment. The predictive algorithm indicated a pooling ratio of 4 in 1 was expected to retain accuracy of the test irrespective of the ct value (relative RNA copy number) of the sample spiked and result in a 237% increase in testing efficiency. Conclusions: The approaches showed its concept in easily customized and resource-saving manner and would allow expanding of current screening capacities and enable the expansion of detection in the community. The coronavirus induced disease 2019 (COVID-19) pandemic has revealed the global public health importance of robust diagnostic testing to differentiate severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) from other routine respiratory infections and to guide appropriate public health and individual clinical management [1] . Detecting carriers of the virus at various population levels is fundamental to response efforts. It ensures the quarantine of COVID-19 patients to prevent local community transmission, and more broadly informs national response team to take measures [2] . However, it remains uncertain whether there may have been community circulation of SARS CoV-2 prior to the identification of individuals with positive results through standard public health surveillance as detection and monitoring capacity is limited [3] , and testing in Ethiopia is generally done on handful of facilities, while potentially infectious carriers at the community remain undiagnosed. Given the limited testing capacity available in Ethiopia, the decision to test is based on clinical and epidemiological factors and linked to an assessment of the likelihood of infection and testing of appropriate specimens from patients meeting the suspected case definition for COVID-19 is a priority for clinical management and outbreak control [3] . Thus, it is necessary to come up with new ways to efficiently and effectively use available resources. Sample pooling (mixing of samples and testing at a single pool, and subsequent individual testing needed only if the pool tests positive) has been used as an attractive method for community monitoring of infectious diseases as it requires no additional training, equipment, or materials [4] [5] [6] . The key principles for successful application of group testing involve knowledge of the limit-of-detection, sensitivity, and specificity of the assay, and the prevalence of disease in a given population (7, 8) . Here we have shown a proof-of-concept for direct clinical sample and NA pooling for the diagnosis of SARS CoV-2 in Ethiopia using the existing assay. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 12, 2020. . https://doi.org/10.1101/2020.06.10.20123398 doi: medRxiv preprint Objective: To establish an efficient, time and resource-saving and cost-effective methods and to propose an ad hoc laboratory-based surveillance approach for mass screening of SARS-CoV-2 With this assay, a positive SARS CoV-2 result is determined when both targets reach a defined Ct value of less than 40, along with defined Ct value of less than 32 and 40 for positive control and internal control, respectively. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 12, 2020. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 12, 2020. . https://doi.org/10.1101/2020.06.10.20123398 doi: medRxiv preprint proportionally to have a final NA volume of 5μL as indicated above in Table 1 . For detecting a single positive sample within a pool of negative nucleic acid extracts, we evaluated the ability of the standard qRT-PCR test under the protocol recommended by manufacture of the kits. Then, change in ct value of samples with low, medium and high ct value was analyzed. To assess the pool testing strategy, the most efficient pool size was calculated using a web based application of pooling (https://www.chrisbilder.com/shiny). As per the key principles of pooling, the following assumptions with numeric parameters are considered: an experimental prevalence rate of SARS CoV-2 in Ethiopia to be 0.05% (as observed positive rate within the tested individuals is reaching to 0.66% in the last 5 weeks), a two-stage pooling in a range of pool sizes 3-10 samples, an assay limit of detection (LOD) of 2.5 RNA copies/μL of reaction, an assay sensitivity of 98% -100% and an assay specificity of 100%. With these calculations, a pool size of 4 samples predicted and would provide the largest reduction in the expected number of tests of 60% when compared to testing clinical samples separately (Table 2 ). . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 12, 2020. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 12, 2020. . https://doi.org/10.1101/2020.06.10.20123398 doi: medRxiv preprint (Figure 2a and 2b) . The results showed that RNA pooled samples were positive within a range of 0 Ct to 3.45 and 0 to 3.19 Ct value difference from the original samples for N and ORF1ab genes, respectively. b . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 12, 2020. CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 12, 2020. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 12, 2020. . https://doi.org/10.1101/2020.06.10.20123398 doi: medRxiv preprint Globally, shortage of molecular laboratories for the diagnosis of SARS CoV-2, lack of trained human capital, lack of NA extraction, amplification and detection kits, and lack of accessory and supplementary consumables despite an increasing number of testing demands has become an issue of concern (8, 9) and nationally in a relatively short period of time. To minimize work load, resources and costs, a pooling approach of NA extractions or amplification and detection might be considered. Here, we showed a proof-of-concept for direct clinical sample and . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 12, 2020. . https://doi.org/10.1101/2020.06.10.20123398 doi: medRxiv preprint RNA/NA pooling for the diagnosis of SARS CoV-2 in Ethiopia using the existing assay. Results from these pooling methods support that pooled screening strategy can be pursued to increase testing throughput, limit use of reagents, and increase overall testing efficiency [6, 7] at an expected slight loss of sensitivity for direct clinical sample pooling. The same could be attained with no loss of sensitivity for RNA pooling. This study also showed whether pooling was feasible using SARS CoV-2 assay in a both public and clinical setting where the desire to test large numbers of individuals has been impacted by the scarcity of key resources in the country. The predictive algorithm indicated a direct clinical sample pooling ratio of 4 in 1 was expected to retain accuracy of the test irrespective of the ct value (relative RNA copy number) of the sample spiked, and results in a 237% increase in testing efficiency. Furthermore, the predictive algorithm indicated an RNA pooling ratio of 10 in 1 was expected to retain accuracy of the test irrespective of the ct value (relative RNA copy number) of the sample spiked, and results in a 140% increase in testing efficiency. The practical application of this process was confirmed in the saving of reagents and personnel time that could expand testing. Assuming a consistent positivity rate in the country, this direct biological and RNA pooling strategy would expand testing by 237% and 140%, respectively. However, in a rapidly changing epidemic, testing strategies will need to adapt to real time potential increases in the prevalence rate of a diseases which also requires the use of highly sensitive assays to avoid missing samples with low RNA copy number [7] [8] [9] . Furthermore, the impact of different extraction methods on the recovery of RNA/NA and overall assay sensitivity need to be evaluated. And, thus both public and clinical laboratories must perform their own validation pool studies for their own methods of RNA/NA extraction and amplification/detection aligning with the prevalence rates of SARS CoV-2 in real time of the settings. Thus, due to the availability of existing limited . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 12, 2020. . https://doi.org/10.1101/2020.06.10.20123398 doi: medRxiv preprint SARS CoV-2 diagnosis facility, access to diagnostic tests, kit supplies, the increasing number of individuals to be tested and available trained human capital, this approach facilitates rational use of resources. Furthermore, the approaches could allow for prospective monitoring the effectiveness of contact reduction measures at the population level and early detection of epidemic waves. However, the limitation of this study is that because of the lack of a plasmid with known concentration, we were not able to quantify the changes occurred in between the dilutions in terms of viral copy number. Considering an increasing SARS CoV-2 epidemic and the possibility of unrecognized spread of the diseases within the community we propose a rapid and straightforward screening strategy for SARS CoV-2 using either direct biological sample polling 4 in 1 or RNA pooling up to 8 in 1. This approach proved its concept and principles, and may facilitate detection of early community transmission of SARS CoV-2 to enable the timely implementation of appropriate infection control measures to reduce spread. The method can also be used for routine monitoring of essential work groups. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 12, 2020. Institute/ALERT Ethics Review Committee has also waived it. Availability of data and materials: All data generated or analysed during this study are included in this article Competing interests: The authors declare that they have no competing interests Funding: None . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 12, 2020. . https://doi.org/10.1101/2020.06.10.20123398 doi: medRxiv preprint World Health Organization Director-General's Opening Remarks at the Media Briefing on COVID-19 -11 Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV2) National Comprehensive COVID-19 Management Handbook Pooling nasopharyngeal/ throat swab specimens to increase testing capacity for influenza viruses by PCR Assessment of Specimen Pooling to Conserve SARS CoV-2 Testing Resources Ad hoc laboratory-based surveillance of SARS-CoV-2 by realtime RT-PCR using minipools of RNA prepared from routine respiratory samples Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Laboratory readiness and response for novel coronavirus (2019-nCoV) in expert laboratories in 30 EU/EEA countries Comparison of nasopharyngeal and oropharyngeal swabs for SARS-CoV-2 detection in 353 patients received tests with both specimens simultaneously